﻿Elachistadimicatella sensu auctt.—a complex of neglected species diversity (Lepidoptera, Elachistidae) from European mountain systems

﻿Abstract Elachistadimicatella Rebel, 1903, has so far been considered a species in Europe with restricted distribution from Ukraine to western France. The species occurs on mountainous regions. However, the in-depth analysis of a taxonomically uncertain species of Elachista from the Cottian Alps (Italy), especially through DNA barcoding and subsequent morphological studies, led to the realization that individuals previously identified as E.dimicatella from the Cottian Alps and the Pyrenees were misidentified. According to our research, a total of three species can be differentiated: E.dimicatella from Carpathians and its former junior synonym E.niphadophanes Meyrick, 1937, sp. rev., from the Pyrenees, as well as the newly described E.cottiellasp. nov. from southwestern Alps, hitherto incorrectly identified as E.dimicatella. Diagnostic features of the three species are discussed and illustrated. Elachistadimicatella and E.niphadophanes are redescribed.


Introduction
Elachistinae is an extremely diverse subfamily of micromoths with more than 800 valid species worldwide placed in 10 genera (Kaila 2019).Furthermore, many undescribed species are known, and further taxa are expected to be discovered from large and virtually unexplored areas, particularly in Asia, Africa, and South America.Conversely, the European fauna is comparatively well explored, although new species are described almost every year, particularly from the Mediterranean.Additionally, the larger mountain systems of Europe, such as the Alps or the Dinarides, are likely to harbor a considerable potential for previously unrecognized species diversity.Therefore, it was not a big surprise that, as part of a comprehensive molecular survey of the regional Lepidoptera fauna in the northern Cottian Alps, several not identifiable species, including an Elachista sp., were discovered (Huemer and Wieser 2023).Morphology and DNA barcodes grouped this taxon as part of the diverse but taxonomically in-ZooKeys 1212: 179-194 (2024), DOI: 10.3897/zookeys.1212.126598 Lauri Kaila & Peter Huemer: Elachista dimicatella sensu auctt.
sufficiently revised Elachista bifasciella group first defined by Traugott-Olsen and Nielsen (1977), which, according to DNA barcoding with an extensive taxon sampling by Mutanen et al. (2013), encompasses a surprisingly large amount of cryptic diversity.However, DNA barcodes of the southwestern alpine taxon were previously unknown.They grouped as the nearest neighbour to E. dimicatella Rebel from the Ukrainian Carpathians, but with a significant genetic distance of about 6%.Subsequent research demonstrated that specimens previously identified as E. dimicatella from the southwestern Alps (Parenti and Varalda 1994) actually belong to the beforementioned morphologically and genetically distinct species, apparently without an available name.Investigations of type material of another taxon synonymized with E. dimicatella from the French Pyrenees, E. niphadophanes Meyrick, finally confirmed a third species within this hitherto overlooked species group.

Materials and methods
The present paper is based on material from the following collections:

MZH
Finnish Museum of Natural History, Helsinki, Finland Material of the new species collected by PH was attracted to artificial light (UV tubes) or bred (Parenti and Varalda 1994).

Dissection
The terminology of genitalia follows the standard work of Traugott-Olsen and Nielsen (1977), with some modifications by Kaila (1999Kaila ( , 2011)).To stain the male genitalia, an aqueous solution of red (yellow) eosin was used.The abdominal pelt of male specimens, and both the abdominal pelt and the genitalia of female specimens, were gently stained using chlorazol black.

DNA barcodes
Tissue samples (dried legs) of three specimens of the new species were prepared for DNA barcoding (Hebert et al. (2003) processed at the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph), following deWaard et al. (2008).In addition, 76 public and private barcode sequences of nine species of Elachista, considered to belong to the Elachista bifasciella species group, in the Barcode of Life Data Systems (BOLD; Ratnasingham and Hebert 2007;Ratnasingham 2018) were used for analysis.All barcodes, except for a single shorter sequence of E. dimicatella, range between 550 and 658 bp.Further details of sequenced material including complete voucher data and images of specimens can be accessed in the public dataset DS-ELACDIMI "Elachista dimicatella species group" https://doi.org/10.5883/DS-ELACDIMI in the Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert 2007;Ratnasingham 2018).Sequences from the dataset were finally submitted to GenBank.Degrees of intra-and interspecific variation of DNA barcode fragments were calculated using the Kimura two-parameter model on the platform of BOLD systems v. 4.0.(https://boldsystems.org).Calculation of intraspecific distance was furthermore normalized with BOLD calculation tools to reduce bias in sampling at the species level.
Sequences were assigned to Barcode Index Numbers (BIN).BINs were automatically calculated for records in BOLD that comply with the DNA Barcode standard (Ratnasingham and Hebert 2013).
A neighbour-joining tree was constructed using the Kimura-2-parameter model in MEGA7 (Kumar et al. 2016).

Photographic documentation
Photographs of adults were taken with an Olympus OM-D Mark III camera and a 60 mm macro lens.Genitalia photographs of E. niphadophanes with a Zeiss Axiolab 5 microscope, mounted with an Olympus OM-D Mark III camera; 60 to 90 stacked photographs were edited using Helicon Focus 4.8 and Adobe Photoshop 6.0.Other genitalia images were taken with a Leica DM4000 B LED camera, and were edited, and the plates of each specimen assembled using several versions of Corel Photo-Paint included in CorelDRAW Graphics suite.Comparison of the length of the phallus in relation to the valva was measured as the longest line from the base of the sacculus to the apex of the cucullus.In the assembled male genital plates the true length of the phallus as compared with other parts of the genitalia is shown with the phallus positioned in the left side of the genitalia.The magnified images of the phallus and the juxta do not reflect their relative sizes among the species.The same applies to the "whole genitalia with phallus" part.These images are not at the same scale between images.The size of the genitalia correlates with the size of the specimen, so the wingspan can be used as a proxy for the relative sizes of the genitalia.

Results of molecular analysis
Molecular analysis is based on 79 DNA barcodes sequences for nine Elachista species belonging to the Elachista bifasciella species group.The interspecific distances to the nearest neighbour in this dataset vary from 4.14% to 8.59% per species pair.Intraspecific barcode variation is distinctly lower but based on only limited material for few species.It exceeds 2% only in E. bifasciella which clusters in 2 BINs, one geographically limited to southern Austria, whereas all other specimens cluster in a single and unique BIN (Table 1, Fig. 1).Diagnosis.Elachista dimicatella is externally characterized by the creamy white head, the white base of the nearly black forewing reminiscent of E. diederichsiella Hering, straight and relatively narrow median fascia, and the usually medially confluent subcostal (tornal) and costal spots, often forming a steep angle towards apex.The antennae of the male are unicolourous grey in the male, in the female paler in apical half; in E. cottiella the antennae of both sexes are unicolourously dark grey, in E. niphadophanes the antennae of the male vary.In the male genitalia E. dimicatella is close to E. niphadophanes from which it differs by the somewhat longer phallus.From E. cottiella it is distinguished by the white pattern of forewings, that being somewhat silvery in E. cottiella.Subcostal and tornal spots are clearly separate in E. cottiella.In the male genitalia the most distinctive difference between E. dimicatella and E. cottiella is the markedly longer phallus in E. cottiella in which the shape of the juxta lobes is more rounded than in the other species.The antrum is significantly longer and narrower in E. cottiella than in E. dimicatella in which it is distinctly convex.Apophyses anteriores are thinner in E. dimicatella than in E. cottiella.More detailed diagnosis between E. dimicatella and E. cottiella are presented in the diagnosis of the latter species.The female of E. niphadophanes is unknown.2, 3).Wingspan 8.5-11 mm, male on average larger than female.Labial palpus ascending, approximately as long as diameter of head, off-white to silvery grey above, fuscous below; 3 rd segment often paler than 2 nd segment.Head creamy white; neck tuft varying from white or grey to almost black, thorax dark grey, tegula in basal half dark grey, white in distal half.Antenna entirely dark grey in male, distally a little paler in female.Fore-and midleg dark grey; hindleg outwards dark grey, inwards off-white with also spurs, tibia and tarsal articles distally off-white.Ground colour of forewing very dark brown to nearly black; base white especially on dorsal side; white transverse fascia of varying width at 1/3 forewing length; similarly coloured subcostal and tornal spot at 3/4 forewing length, confluent forming medially outward directed fascia, in female sometimes separate; fringe as ground colour, at termen white.Underside of fore-and hindwing dark grey with concolourous fringe.

Redescription. Habitus (Figs
Male genitalia (Fig. 7).Uncus lobes widely apart from each other, separated by convex posterior margin of tegumen, ventrolaterally directed, tongue-shaped, 2 × as long as wide, distally round.Spinose knob of gnathos small as compared to average size within the E. bifasciella group.Valva slightly bent, broadest medially, 3.6 × as long as wide at its widest part medially; basal fold of costa extended to distal 3/4 of valva where it meets distal fold forming indistinct hump.Digitate process ¼ as long as valva, narrow and parallel-sided, with a few setae distally.Mesial margin of juxta lobes straight, meeting distal margin at a right angle, distal margin nearly straight, broadly setose.Vinculum tapered, distally v-shaped, no distinct median ridge.Phallus 0.75 × as long as valva, straight, distal end bifurcate; without cornuti; caecum short, round, posterior opening dorsally projected.
Female genitalia (Fig. 10).Papilla analis round in lateral view; membrane between papillae anales ventrally densely covered by minute spines, ventral margin otherwise with row of setae, setae longest at apex of papilla analis.Apophysis posterioris as long as papilla analis.Apophysis anterioris 2/3 × as long as apophysis posterioris.Ostium bursae in anterior margin of tergum 8, posterior margin convex; dorsal wall spinose; length of antrum slightly less than length of apophysis posterioris; antrum convex, colliculum posteriorly more and anteriorly less sclerotized, length as measured from anterior end of antrum to inception of ductus seminalis almost 3 × as long as apophysis posterioris; ductus bursae otherwise tubular, membranous, 1.5 × as long as antrum + colliculum, widened anteriorly, incepted in corpus bursae without clear limit; corpus bursae round, with small internal granules; signum elongate, dentate, broadest medially.
Biology.In the Polish Tatra Mountains the larvae were collected in late May and June, preferably feeding on Sesleria tatrae and Deschampsia caespitosa, and furthermore on Alopecurus pratensis, Calamagrostis arundinacea, C. villosa, Dactylis glomerata, Milium effusum, and Poa alpina.Adults were observed from late June to July.The habitat is described as grassland on sunny slopes, exclusively on calcareous soil at altitudes between 1000 and 1800 m (Buszko and Baraniak 1989).Steuer (1976) gives a detailed description of the larva habits as leaf-miners in Sesleria coerulea.
Several additional host-plants published by Parenti and Varalda (1994) cannot be attributed to a host-species and require confirmation.

Elachista cottiella
Diagnosis.Elachista cottiella differs from the closely related E. dimicatella and E. niphadophanes by the entirely grey antennae in both male and female, entirely grey tegulae, a narrower silvery-white medial fascia and smaller and separate subcostal and tornal spots of the forewing, and entirely dark fringe.In the former mentioned species, wing markings are broader with costal and tornal spots medially fused, and the tip of forewing fringe is white.The digitate process is shorter in E. cottiella and E. niphadophanes than in E. dimicatella.The lateral margins of the juxta lobes are more convex in E. niphadophanes than in E. cottiella and E. dimicatella thus making it more round than in the other species.The phallus of E. niphadophanes is shorter than in either other species, 0.6 × as long as valva, basally bulbous.The antrum is significantly longer and narrower in E. cottiella than in E. dimicatella in which it is distinctly convex.Apophyses anteriores are stouter in E. cottiella than in E. dimicatella.The female of E. niphadophanes is unknown.
Description.Habitus (Figs 5,6).Wingspan 6.5-8.5 mm (reared specimens), male on average larger than female.Labial palpus ascending, approximately as long as diameter of head, off-white to silvery grey above, broadly dark grey below.Head off-white to creamy, shiny; neck tuft varying from creamy to grey; thorax dark grey, tegula grey.Antenna entirely grey both in male and female.Legs shiny grey; hindleg inwards off-white with also spurs, tibia and tarsal articles distally offwhite.Ground colour of forewing very dark brown to nearly black; base sometimes, in female in particular, silvery white on dorsal side; white or silvery, rather narrow transverse fascia of at 1/3 forewing length, metallic shine more pronounced in female; similarly coloured subcostal and tornal spot near apex of forewing separate, subcostal spot closer to apex than dorsal spot; fringe as ground colour.Underside of fore-and hindwing dark grey with concolourous fringe.
Male genitalia (Fig. 9).Uncus lobes widely apart from each other, separated by convex posterior margin of tegumen, ventrolaterally directed, tongueshaped, 2 × as long as wide, distally round.Spinose knob of gnathos small as compared to average size within the E. bifasciella group, longer than wide.Valva straight or slightly bent, basal fold of costa extended to distal 3/4 of valva where meeting distal lobe and forming variably distinct hump.Digitate process 1/5 × as long as valva, parallel-sided, with a few setae distally.Mesial margin of juxta lobes slightly bent to median direction, meeting distal margin at a right angle, distal margin somewhat concave, laterally setose, lateral margin distinctly concave.Vinculum tapered, distally v-shaped, with indistinct median ridge.Phallus as long as valva, broadest in basal 1/4, straight, distal end bifurcate; without cornuti; caecum short, rounded, posterior opening dorsally projected.
Biology.Host-plants insufficiently documented.All reared specimens available to us have been reared from Avenella flexuosa and Festuca sp. in rubra group.Several host-plants from the Polish Tatra Mountains attributed to the former senior synonym E. dimicatella are not applicable for the new species.However, additional host-plants published by Parenti and Varalda (1994) could all belong to E. cottiella.Elachista cottiella was found on siliceous soil in montane to subalpine open grassland with adults attracted to UV lamps (Fig. 12).
Etymology.The new species name refers to the known distribution area.

Discussion
The documentation of the diversity of European Elachista species has advanced considerably, not in the least due to the increasing implementation of molecular methods.The status of described species is still largely supported by the study of historical type material (Kaila 2019), but increasingly there are more publicly available DNA barcode data.Nevertheless, despite these advancements, the presence of undescribed species remains a significant challenge.The unexpected discovery of a previously overlooked or taxonomically misinterpreted species complex, as highlighted by Mutanen et al.'s (2013) extensive molecular investigations in this group, adds an element of surprise.It appears that the expanded geographical coverage within this family suggests the likelihood of further hidden diversity.
For instance, Huemer (2020) and Huemer and Wieser (2023) have identified additional cryptic species from the Alps lacking reference sequences in BOLD.
The taxa now revised, and previously grouped under the name E. dimicatella, exhibit small but diagnostic differences in phenotype, genital morphology, and molecular characteristics (DNA barcode).Notably, all these species have been observed exclusively in allopatry, a pattern observed in many butterflies and moths across European mountainous regions (Fig. 13).The substantial variations in species compositions among genera of montane to alpine Lepidoptera in the Pyrenees, Alps, and Carpathians are well documented (Huemer and Tarmann 1992;Whitebread 2006;Huemer and Hausmann 2009;Huemer and Hebert 2011;Zlatkov and Huemer 2017).